BACKGROUND This study aimed to compare amino acid concentrations in pregnant patients with obesity and gestational diabetes mellitus (GDM) to those in healthy pregnant women at different stages of pregnancy. MATERIAL AND METHODS The study group consisted of 50 pregnant women with obesity (body mass index [BMI] >30 kg/m²) diagnosed with GDM in the first half of pregnancy. The control group included 25 healthy pregnant women with a BMI <25 kg/m². Blood samples were collected at visit V1 (no later than 20 weeks of gestation) and V2 (between weeks 37 and 39). The amino acid profiles were determined using a hybrid triple quadrupole/linear ion trap mass spectrometer coupled with a liquid chromatography instrument. RESULTS Of the 30 measured amino acids, 23 serum concentrations differed significantly in patients with obesity. Proline (P<0.001), leucine (P=0.001), valine (P=0.001), and isoleucine (P=0.001) were higher in the obesity group than the control group. Conversely, glutamine (P=0.001), taurine (P=0.008), and asparagine (P=0.001) were significantly lower in obesity group than control group during both observation periods. Changes in isoleucine (P=0.023), tyrosine (P=0.011), taurine (0.032), and phenylalanine (P=0.009) concentrations were associated with patients' weight gain during pregnancy. CONCLUSIONS Research on amino acids offers a detailed understanding of the metabolic interactions that play a crucial role. Metabolomics has emerged as a technology with significant potential for early detection of GDM and for enhancing our understanding of the pathogenesis and impact of the disease on both the mother and her offspring.